Figure 5

RNase R regulates SmpB but not tmRNA levels. Northern blot and Western blot analysis of RNA and protein samples extracted from wild type and mutant strains as indicated on top of each lane. Details of experimental procedures are described in ‘Methods’. (a) Analysis of tmRNA by Northern blot. 15 μg of RNA extracted from the wild type (WT) and the RNase R^- mutant at 15°C and 37°C were separated on a 6 % polyacrylamide/8.3M urea gel. The gel was then blotted to a Hybond-N⁺ membrane and hybridized with a tmRNA specific riboprobe. (b) Analysis of SmpB protein (~18 kDa) and mRNA levels. (Upper panel) 15 μg of total RNA extracted in the same conditions from the wild type, the RNase R^- mutant and the RNase R^- strain expressing RNase R from pIL253, were separated on a 1.5 % agarose gel, transferred to a Hybond-N⁺ membrane and hybridised with a specific probe for smpB. The membrane was stripped and then probed for 16S rRNA as loading control. (Lower panel) SmpB protein levels were analysed by Western blot with SmpB specific antibodies. 20 μg of total protein samples extracted in the same conditions were separated in a 10 % tricine-SDS polyacrylamide gel and blotted to a nitrocellulose membrane. A non-specific band (Control) detected with the same antibodies was used as loading control.